medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Phylogeography and Resistome Epidemiology of Gram-Negative Bacteria in Africa: A

Systematic Review and Genomic Meta-Analysis from a One-Health Perspective

John Osei Sekyere#1 and Melese Abate Reta1

Department of Medical Microbiology, School of Medicine, Faculty of Health Sciences,

University of Pretoria, South Africa1

#Address correspondence to: Dr. John Osei Sekyere, Department of Medical Microbiology,

School of Medicine, Faculty of Health Sciences, University of Pretoria, South Africa. Email:

[email protected]; [email protected] Tel. +27- (0)12-319-2251

Running head: Epidemiology of Gram-negative bacterial resistance, Africa

Highlights/Significance

Antibiotic resistance (AR) is one of the major public health threats and challenges to effective

containment and treatment of infectious bacterial diseases worldwide. Herein, we used

different methods to map out the geographical hotspots, sources and evolutionary

epidemiology of AR. Escherichia coli, Klebsiella pneumoniae, Salmonella enterica,

Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter spp., Neisseria

meningitis/gonnorhoeae, Vibrio cholerae, Campylobacter jejuni etc. were common

pathogens shuttling AR genes. Transmission of same clones/strains across countries and

between animals, humans, plants and the environment were observed. We recommend

Enterobacter spp. or K. pneumoniae as better model/index organisms for AR surveillance.

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Abstract

Objectives/Backgrounds: Antibiotic resistance (ABR) remains a major threat to public

health and infectious disease management globally. However, ABR ramifications in

developing countries is worsened by limited molecular diagnostics, expensive therapeutics,

inadequate skilled clinicians and scientists, and unsanitary environments.

Methods: Data on specimens, species, clones, resistance genes, mobile genetic elements, and

diagnostics were extracted and analysed from English articles published between 2015 and

December 2019. The genomes and resistomes of the various species, obtained from PATRIC

and NCBI, were analysed phylogenetically using RAxML and annotated with Figtree. The

phylogeography of resistant clones/clades was mapped manually.

Results & conclusion: Thirty species from 31 countries and 24 genera from 41 countries

were respectively analysed from 146 articles and 3028 genomes. Genes mediating resistance

to β-lactams (including blaTEM-1, blaCTX-M, blaNDM, blaIMP, blaVIM, blaOXA-48/181),

fluoroquinolones (oqxAB, qnrA/B/D/S, gyrA/B and parCE mutations etc.), aminoglycosides

(including armA, rmtC/F), sulphonamides (sul-1/2/3), trimethoprim (dfrA), tetracycline

(tet(A/B/C/D/G/O/M/39)), colistin (mcr-1), phenicols (catA/B, cmlA), and fosfomycin (fosA)

were mostly found in Enterobacter spp. and K. pneumoniae, and also in Serratia marcescens,

Escherichia coli, Salmonella enterica, Pseudomonas, Acinetobacter baumannii, etc. on

mostly IncF-type, IncX3/4, ColRNAI, and IncR plasmids, within IntI1 gene cassettes,

insertion sequences and transposons. Clonal and multiclonal outbreaks and dissemination of

resistance genes across species, countries and between humans, animals, plants and the

environment were observed; E. coli ST103, K. pneumoniae ST101, S. enterica ST1/2 and V.

cholerae ST69/515 were common strains. Most pathogens were of human origin and

zoonotic transmissions were relatively limited. One Health studies in Africa are needed.

[2] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Keywords: One Health; Antibiotic resistance; molecular epidemiology; diagnostics;

genomics; resistome; mobilome; Africa

Introduction

Antibiotic resistance (ABR), particularly in Gram-Negative bacteria (GNB), is complicating

infection management in Africa and the rest of the world as it restricts effective therapeutic

options available to clinicians in human and veterinary medicine 1–3. Given the unsanitary

environments common in developing countries as well as the limited healthcare and

laboratory facilities, poor sewage management, high patient-to-physician ratios, little or no

regulation on antibiotic usage etc., the escalation of ABR in developing countries (Africa) is

precarious 3–5. Coupled with these is the limited funding available for molecular surveillance

of ABR to map out the true burden of the problem 2,4. A recent global metagenomic survey,

for instance, found that African countries had the highest ABR genes (ARGs) abundance,

although in Animals, Asia was found to have more ABR hotspots than Africa 2,5.

Together, GNB cause or aggravate some of the most fatal and common infections and

diseases known to mankind: sepsis, meningitis/meningococcemia, gonorrhoea, pneumonia,

cystic fibrosis, urethritis, pelvic inflammatory disease, cholera, typhoid, whooping cough,

diarrhoea etc6–15. It is worth noting that compared to Gram-positive bacteria, GNB has been

implicated in the development of more resistance mechanisms, including resistance to last-

resort antibiotics such as colistin (mcr), carbapenems (blaNDM, blaVIM, blaKPC, blaIMP, blaGES

etc.) and tigecycline (tet(X)) 1,16,17. Moreover, these ARGs are found in more diverse GNB

species and genera, with substantial mortalities, morbidities and multi-drug resistance 16–19.

Hence, GNB species such as Pseudomonas aeruginosa, Acinetobacter baumannii, and

Enterobacteriaceae expressing resistance to carbapenems and extended-spectrum β-

lactamases (ESBLs) are respectively classified as critical and high priority pathogens by the

WHO 20,21.

[3] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

The presence of these ARGs and species in human, environmental and animal specimens in

Africa (and globally) are well documented, strengthening the call for further One Health

molecular surveillance 2,3,5,22. Yet, a systematic review on GNB in Africa from a One Health

perspective is lacking 22,23. This limits the comprehensive appreciation of the epidemiology of

ARGs across the continent. Herein, we further undertake a detailed phylogenomic,

phylogeographic and resistome analyses using publicly available genomes to augment the

findings in the literature and describe the evolutionary epidemiology of ARGs in Africa.

Methods

Databases and Search Strategy

A comprehensive literature search was carried out on PubMed and/or ResearchGate,

ScienceDirect and Web of Science electronic databases. English research articles published

between January 2015 and December 2019 were retrieved and screened using the following

search terms and/or phrases: "molecular epidemiology", "gram-negative bacteria";

"mechanisms of resistance"; antimicrobial resistance genotypes"; "drug resistance"; "AMR

genotypes"; "genetic diversity"; "clones"; "genotyping"; "antibiotic resistance gene";

"plasmid"; "mobile genetic elements"; "resistome"; "gene mutation"; "frequency of resistance

gene mutation"; and Africa. The search terms used were further paired with each other or

combined with the name of each African country, and searching strings were implemented

using "OR" and "AND" Boolean operators.

Inclusion and Exclusion Criteria

Articles addressing the molecular mechanisms (using PCR, microarray or WGS) of ABR in

GNB and undertaking bacterial typing (MLST, PFGE, ERIC-PCR, WGS etc.) were included

in this systematic review. Papers which addressed ABR using only disc diffusion, broth

microdilution, E-test, Vitek, MicroScan etc. without a molecular (PCR/microarray/WGS)

[4] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

analyses of the underlying resistance mechanism and typing (clonality) of the isolates were

excluded.

Included data

This review was carried out using the Preferred Reporting Items for Systematic Reviews and

Meta-Analyses (PRISMA) guidelines (Figure 1). The following data were extracted from the

included articles: country; study year; specimen type and source/s; sample size; total isolates

(isolation rate); total isolates for which antimicrobial sensitivity testing (AST) was

determined; bacterial species; clone/MLST; antibiotic resistance gene (ARGs); mobile

genetic elements (MGEs); antibiotic resistance phenotype; genotyping and diagnostic

method(s)/techniques used (Tables S1-S3).

Statistical and bio-informatic analyses

Microsoft Excel® 365 was used to analyse the frequencies using raw data extracted from all

included articles. To each GNB species, the resistance rates per antibiotic per country were

calculated by dividing the total resistant isolates by the total isolates for which antibiotic

sensitivity was determined. Frequencies of resistant species, clones, ARGs-MGEs

associations and ABR rates were evaluated per animal, human and environmental sources per

country (Tables S1-S3).

Genomes of 24 genera that were found in the included articles were downloaded from

PATRIC (https://www.patricbrc.org) and used for phylogenetic analyses using RAxML’s

maximum-likelihood method and annotated with Figtree

(http://tree.bio.ed.ac.uk/software/figtree/) (Table S4); genomes that did not have more than

1000 proteins matching with all the genomes were removed. Default parameters were used to

run the phylogenetic reconstruction with 1000x bootstrap resampling analyses. The resistome

of these genomes were curated from the Isolates Browser database of NCBI

[5] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(https://www.ncbi.nlm.nih.gov/pathogens/isolates#/search/) (Tables S5-S6). The

phylogeography of the resistant clones/clades per species was mapped manually unto an

African map to show their geographical distribution.

Results

Characteristics of Included Studies

The literature search returned 1495 research articles: 1490 articles (from PubMed, Web of

Science, ScienceDirect) and five articles from other sources obtained through manual search.

Duplicates were removed and the remaining 868 non-duplicated articles’ titles and abstracts

were screened. Full-text review of the remaining 309 manuscripts resulted in 146 studies

being used for the qualitative and quantitative analyses (Figure 1) (Tables S1-S3).

The included articles spanned 31 countries from animal (A), human (H) and environmental

(E) samples, with South Africa having the most (n=24) studies: Algeria (n=10; A=3, H=6,

E=1); Benin (n=2; H=1, E=1); Botswana (n=2; A=1, H=1); Burkina Faso (n=7; A=1, H=

6,); Cameroon (n=3; A=1, H=2); Central African Republic (CAR)(n=2; H=2); Chad (n=1;

H=1); Cote D’Ivoire (n=1; H=1); Democratic Republic of Congo (DRC) (n=2; H=1, E=1);

Egypt (n=18; A=5, H=12, E=1); Ethiopia (n=5; A=2, H=3); Gabon (n=1; A=1); Ghana

(n=5; A=1, H=4); Kenya (n=6; A=1, H=5); Libya (n=2; H=2); Malawi (n=1; H=1); Mali

(n=1; H=1); Morocco (n=1; H=1); Mozambique (n=1; H=1); Niger (n=2; H=2); Nigeria

(n=15; A=1, H=9, E=5); Sao Tome & Principe (n=1; H=1); Senegal (n=2; H=2); Sierra

Leone (n=2; H=2); South Africa (n=35; A=4, H=24, E=7); Tanzania (n=10; A=3, H=6,

E=1); Tunisia (n=10; A=3, H=7); Uganda (n=2; A=1, H=1); Zambia (n=2; A=1, H=1);

Zimbabwe (n=2; A=1, H=1).

These studies involved 23,157 isolates from 65,119 specimens (isolation rate of 35.56%):

2,560 isolates from 5,950 animal specimens (43.03% isolation rate), 16,225 isolates from

[6] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

57,464 human specimens (isolation rate of 28.24%), and 4,372 isolates from 1,705

environmental specimens (isolation rate of 256.42%). The various species identified per each

study is summarised under the respective specimen source in Table 1; the per country

breakdown is shown in Tables S1-S3.

The 3028 genomes included in this study were also obtained from animals, humans, plants

and the environment from 41 African countries: Angola, Benin, Botswana, Burkina Faso,

Cameroon, CAR, Chad, Comoros, DRC, Djibouti, Egypt, Eritrea, Ethiopia, Gambia, Ghana,

Guinea, Guinea-Bissau, Kenya, Lesotho, Madagascar, Malawi, Mali, Mauritania, Mauritius,

Morocco, Mozambique, Namibia, Niger, Nigeria, Republic of the Congo, Rwanda, Senegal,

Sierra Leone, South Africa, Sudan, Tanzania, Togo, Tunisia, Uganda, Zambia, and

Zimbabwe (Table S6; Figures 2-8; Figures S7-S25).

Species distribution (from included articles)

Of the 30 species isolated from the various human, animal and environmental specimens

included in the studies used for this meta-analysis, the commonest were Escherichia spp.

(n=9292), Klebsiella spp. (n=2776), Salmonella enterica (n=1773), Pseudomonas spp.

(n=1498), and Acinetobacter spp. (n=705), which were all commonly isolated from human

samples than from animal or environmental specimens; these statistics were also largely

reflected in the species distribution in the genomics data (Table 1). These common

pathogens, including Neisseria gonorrhoea/meningitidis, Proteus mirabilis and Enterobacter

spp., were mostly concentrated in Algeria, Burkina Faso, Egypt, Ghana, Kenya, Libya, South

Africa, Tanzania and Tunisia in humans (Fig. S1). South Africa, Tanzania and Nigeria,

reported the highest concentrations of environmental species. Notably, E. coli and S. enterica,

and to a lesser extent Campylobacter coli/jejuni, Klebsiella spp., and Pseudomonas spp.,

were the most isolated species from animals in the reporting countries. It is interesting to note

[7] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

that N. gonorrhoea/meningitidis were mainly reported from humans in Kenya and Niger

whilst Vibrio cholerae/spp. were mostly isolated from the environment in South Africa and to

a lesser extent, from humans in Cameroon (Fig. S1); yet, genomes of N. meningitidis were

obtained from 10 countries in Southern, Eastern, Western and Northern Africa (Fig. 8).

E. coli was isolated in very high frequencies in almost all reporting countries except Kenya,

Ethiopia, Botswana, Zambia and Senegal (in humans), Ghana, Burkina Faso and Botswana

(in Animals) and Nigeria, Egypt and Cameroon (in the environment). K. pneumoniae was less

common in humans in Egypt, Ethiopia, DRC, Cameroon, Botswana, Benin, Zimbabwe,

Zambia, Niger and Malawi; it was hardly reported from animals in Egypt and Cameroon and

only found in the environment in Nigeria. S. enterica was mainly distributed in Algeria,

Ethiopia, Ghana, Kenya, and Zambia (in humans), and Zambia, Tunisia, S. Africa, Kenya,

Ethiopia and Algeria (in animals); it was only reported from Egypt from the environment. In

humans, P. aeruginosa was mostly found in Egypt, B. Faso, Tanzania, S. Africa and Nigeria

whilst Egypt alone reported it in animals and Nigeria alone reported Pseudomonas spp. in the

environment. Interestingly, A. baumannii was mainly concentrated in Ethiopia and Egypt

(humans), and in Algeria (animals); A. calcoaceticus/spp. was found from the environment in

South Africa and Nigeria (Fig. S1-S2).

Only Campylobacter coli/jejuni had more animal sources (n=210) than human (n=175) and

environmental (n=105) sources and S. enterica was the second most isolated species from

animal specimens after E. coli (Fig. S2). Notably, Vibrio cholerae (n=279),

Stenotrophomonas maltophilia (n=126), Bacillus spp. (n=97), Alcaligenes faecalis (n=47),

Aeromonas spp. (n=11), Chromobacterium spp. (n=6), Brevundimonas spp. (n=3),

Psychrobacter spp. (n=3), Myroides spp. (n=2), and Trabusiella spp (n=1) were either mainly

or only found from environmental sources (Table 1).

[8] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Neisseria meningitidis/gonorrhoea and Mycoplasma genitalium, two sexually transmitted

infectious pathogenic species, were mainly found in clinical samples (Table 1; Fig 5).

However, other Mycoplasma spp. were found in chicken (M.

gallinarum/gallinaceum/pullorum), goat (M. bovis), cattle (M. mycoides), ostrich (M.

nasistruthionis), sewage (M. arginini) and humans (M. pneumoniae), and these had no known

resistance genes (Fig. S19). Species such as Burkholderia cepacia/spp., Bordetella spp.,

Morganella morganii, A. faecalis, S. maltophilia, Leclercia adecarboxylata, Pantoea spp.

and Raoultella spp. were rare in clinical samples (Table 1; Fig. S14, S20 & S24).

Geographical and host distribution of clones, ARGs and MGEs

The clonality of A. baumannii, C. coli/jejuni, E. coli, K. pneumoniae, P. aeruginosa, V.

cholerae, S. marcescens, N. gonorrhoeae/meningitidis, and S. Entiritidis/Typhi/Typhimurium

strains were reported from the various countries out of the 30 species (Fig. S3). However,

only E. coli clones viz., ST38, ST69, ST131, ST410 etc. and groups A/B/C/D were found in

humans (in Algeria, B. Faso, CAR, Egypt, Libya, Nigeria, Sao Tome & Principe, Tanzania,

Tunisia and Zimbabwe), animals (Algeria, Egypt, Ghana, Tunisia and Uganda) and the

environment (Algeria and South Africa). Specifically, E. coli ST38 was found in humans

(Algeria) and animals (Ghana) and groups A/B/D were found in humans (Egypt), animals

(Algeria, Egypt, Tunisia, Uganda and Zimbabwe) and the environment (Algeria and South

Africa) (Fig. S3). Inter-country detection of E. coli ST131 in humans was also observed in

Algeria, B. Faso, CAR, DRC, Tanzania, Tunisia and Zimbabwe. K. pneumoniae ST101 was

also found in Algeria, South Africa, and Tunisia. As well, multiclonal C. jejuni strains (i.e.

ST19, ST440, ST638, ST9024 etc.) were found in humans and animals from Botswana (Fig.

S3).

The clones of the various species from the genomic data did not always agree with that

obtained from the included articles in terms of geographical distribution and incidence (Fig.

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S3 & Table S6). For instance, the E. coli genomes were highly multiclonal, consisting of 202

clones; the commonest of these were ST661 (n=56), ST10 (n=42), ST443 (n=35), ST131

(n=24), and ST29 (n=10). K. pneumoniae (85 clones) and S. enterica (66 clones) genomes

were also very multiclonal, with K. pneumoniae ST101 (n=40), ST152 (n=22), ST15 (n=16),

ST14 (n=12), ST17 (n=12), ST147 (n=10) and S. enterica ST2 (n=148), ST1 (n=137), ST198

(n=20), ST11 (n=12), ST313 (n=12), ST321 (n=10) and ST2235 (n=10) being very common.

Notably, N. meningitidis (genomes) ST11 (n=45), ST2859 (n=22), ST1 (n=9), ST2859 (n=9)

etc. were also common in humans from Ghana (n=63 isolates), B. Faso (n=57 isolates), Niger

(n=28 isolates) etc. as seen in the articles (Fig. S3 & Table S6). Contrarily, P. aeruginosa

(ST234 & ST235), A. baumannii (ST1, ST85 & ST164), C. jejuni (ST362), V. cholerae

(ST69 & ST515), Bordetalla pertussis (ST1 & ST2 in Kenya), Mycoplasma pneumoniae (in

Egypt and Kenya), Bacillus cereus and Bacillus subtilis (genomes) had relatively few

dominant clones (Table S6).

ARGs mediating resistance to almost all known Gram-negative bacterial antibiotics were

found in the included articles, with most of these ARGs being isolated from human strains

than animal and environmental species in a descending order (Fig. S4). Notably, ARGs

conferring resistance to β-lactams, specifically ESBLs such as CTX-M, TEM, SHV, OXA,

GES and AmpCs such as CMY, FOX, DHA, FOX, MOX, ACC, EBC and LEN were

commonly identified in human, animal and environmental isolates from most countries, with

blaCTX-M and blaTEM being the most identified ARGs (Fig. S2 & S4). Notably, OXA and GES

ESBLs and all the AmpCs as well as carbapenemases (i.e., OXA-48/181/204, OXA-23/51/53,

NDM, IMP, SPM, VIM, KPC, GES-5) were not reported from animal or environmental

isolates; only OXA-61 (from C. jejuni) was found in animal isolates in Botswana.

Carbapenemase genes were relatively less detected in human strains and reported from a few

countries: the metallo-β-lactamases such as NDM, IMP, SPM and VIM were mainly found in

[10] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Egypt, South Africa, Tanzania, Tunisia, and Uganda; KPC and GES-5 were common in

South Africa and Uganda; and the OXA-types were found in Algeria, Egypt, Nigeria, Sao

Tome & Principe, South Africa, Tunisia and Uganda (Fig. S4).

Second to the β-lactams, there were frequent detection of diverse fluoroquinolone resistance

mechanisms in human and animal isolates from almost all the countries: aac(6’)-Ib-cr,

aac(3’)-IIa, aac(3’)-Ih, QnrA/B/D/S, OqxAB, chromosomal mutations in gyrAB and parCE

and qepA in a descending order of frequency. None of these mechanisms were found in

environmental strains. Moreover, aminoglycosides resistance mechanisms, including aac(6’)-

Ib-cr that also confers resistance to fluoroquinolones, were equally highly distributed in

human and animal isolates, with relatively limited occurrence in environmental strains.

Among these aminoglycoside mechanisms were aadA, strAB, aph(3’), aph(6’), ant(2’),

ant(3’), and the 16S rRNA methyltransferases such as rmtC/F and armA.

Other common resistance mechanisms that were highly distributed in almost all strains from

almost all the included countries were sul1/2/3 and dfrA (mediating resistance to sulpha-

methoxazole-trimethoprim); these were mostly found in animal and human strains and

relatively less isolated in environmental strains. Chloramphenicol resistance genes viz.,

cmlA/B and catA/B, were also found in animal and human isolates in substantial numbers

whilst ARGs for florfenicol (floR) and fosfomycin (fosA) were very rare, being found in only

human strains. Of note, tetracycline ARGs, tet(O/A/B/C/D/G/K/M39), were almost fairly

distributed in strains from humans, animals and the environment in relatively few countries

such as Algeria (E), Botswana (A & H), Cameroon (E), Ethiopia (A), Malawi (H), Nigeria

(E), South Africa (A, H, E), Tanzania (A, E), Tunisia (A, H), Uganda (A), and Zambia (A).

Interestingly, colistin resistance mechanisms such as mcr-1 and chromosomal mutations in

pmrAB were very rare. Particularly, pmrAB mutations were only recorded in human strains

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from Tunisia whilst mcr-1 genes were only reported in South Africa (A, H, E), Sao Tome &

Principe (H), and Tunisia (A). Other rare ARGs, found mainly in human isolates, included

blaZ, pse-1, and penA (conferring penicillin resistance), ermABC and mph(A) (encodes

erythromycin/macrolides resistance), cmeAB (multidrug efflux system in Campylobacter

jejuni), porAB (porin in Neisseria spp.), macA/B (encodes part of the tripartite efflux system

MacAB-TolC for transporting macrolides from the cytosol), qacEΔ1 (encodes resistance to

quarternary ammonium compounds through efflux), and mexAB (encodes multidrug

resistance (MDR) efflux pumps in Pseudomonas spp.).

These ARGs were found associated with MGEs such as plasmids, integrons, insertion

sequences (ISs) and transposons, mainly in human isolates; MGEs in animal and

environmental isolates were rarely described (Fig. S5-S6). The commonest MGEs were IncF-

type, IncX3, ColRNAI, IncR, IncY, IncL/M, A/C, IncH, and IncQ plasmids, ISEcP1 and class

1 integrons (IntI1). ColE, IncU, IncLVPK, IncP, IncI, IncN, ColpVc, and ColKp3 plasmids,

Tn2006, ISKpn19, Tn3, IS26, Tn21 and ISAbaI transposons and ISs were less identified

MGEs. Specifically, IncF-type plasmids were commonly associated with ESBLs and

carbapenemase genes whilst IntI1 was common with both β-lactamases and non-β-lactamase

genes such as sul-1/2/3, dfrA, catA/B, floR, qepA and qnrA/B/D/S. ISEcP1 was common

around blaCTX-M-15, Tn206 bracketed blaOXA-23/51 and aac(3’)-I, and Tn402-lik transposon

harboured blaVIM-5, aph(3’)-Ib, aph(6’)-Id, tet(C/G), and floR (in environmental strains in

Nigeria) (Fig. S5-S6).

Resistance rates

Resistance rates to the various antibiotics were highest among human strains, particularly in

Egypt, Ethiopia, Mali, Senegal, Tunisia and Uganda among Enterobacteriaceae such as E.

coli, K. pneumoniae, Salmonella enterica, and Providencia rettgeri/stuartii, Neisseria

meningitidis/gonorrhoea, P. aeruginosa, and A. baumannii. These human strains expressed

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very high resistance to almost all the antibiotic classes including the aminoglycosides, β-

lactams, fluoroquinolones, tetracyclines, sulphamethoxazole-trimethoprim (SXT) and

phenicols. Comparatively, strains from animals and the environment expressed lesser

resistance rates. Specifically, A. baumannii, E. coli, Salmonella enterica, and Providencia

spp., were resistant to ampicillin, amikacin, chloramphenicol, kanamycin, tetracycline,

streptomycin, sulfonamide and SXT in most of the included countries. Notably, the resistance

rates of environmental E. coli, S. enterica, K. pneumoniae/oxytoca, and Citrobacter

freundii/koseri strains to fluoroquinolones, tetracycline, sulphonamide, SXT, and ceftriaxone

were substantially high in Algeria, Benin and Egypt (Tables S1-S3).

Phylogenomic and resistome analyses: evolutionary epidemiology of resistance

Phylogenetically, strains belonging to the same clades were found in different countries, and

in a limited measure, in humans, animals, the environment and in plants. Among the species,

certain countries contained only a single clade of a species whilst some countries contained

several clades of the same species: E coli (Algeria), K. pneumoniae (Mali), N. meningitis

(South Africa, DRC), Campylobacter spp. (South Africa) etc (Fig. 3-8, S7-S25). Within

specific clades were found, in a few cases, isolates from different sample sources: clades A, B

and C (E. coli), clade B (P. aeruginosa) etc. (Fig. 3 & 6, S7). Notably, strains belonging to

different MLSTs were found within the same clades. Generally, the genomes of included

species were from Southern, Eastern, Western and Northern Africa, with little or none from

Central Africa; countries reporting most genomes included Ghana, Mali, Nigeria, Cameroon,

Tunisia, Algeria, Egypt, Kenya, Tanzania, Mozambique and South Africa (Fig. 3-8, S7-S25).

Enterobacter spp. and K pneumoniae strains contained the largest and richest repertoire of

resistomes (Fig. 4, S8 & S17). In a descending order, S. marcescens, S. enterica, E. coli, A.

baumannii, P. aeruginosa, V. cholerae, Citrobacter freundii, Providentia rettgerii, Proteus

mirabilis and M. morganii strains harboured a rich and diverse repertoire of ARGs. No

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known ARGs were found in the other species, although the literature reported resistance

mechanisms in N. meningitidis (gyrA, penA, and rpoB), N. gonorrhoeae (gyrA, penA, ponA,

mtrR, porB and blaTEM), C. jejuni/coli (blaOXA-61, tet(O), gyrA(T86I), cmeABC, and aph(3)-I),

M. genitalium (gyrA and parC) and S. maltophilia (sul3) (Tables S1-S3). Some ARGs were

conserved within specific clades and species: NDM-1 (S. marcescens), OXA-23/66-like (A.

baumannii), TEM -1 (E. coli, K. pneumoniae), CTX-M (Enterobacter spp. and K.

pneumoniae), SUL-1/2/3 (Enterobacter spp., E. coli, K. pneumoniae, S. enterica), dfrA

(Enterobacter spp., E. coli, K. pneumoniae, S. enterica), QnrB/S (Enterobacter spp., K.

pneumoniae), TET (Enterobacter spp., E. coli, N. meningitidis, K. pneumoniae, S. enterica)

etc. (Fig. 3-8, S7-S25).

Diagnostics

Phenotypic and molecular methods were employed by the included studies to determine the

species identity, antibiotic sensitivity (AST), genotype/clone and resistance mechanisms of

the isolates. Broth microdilution (BMD) and disc diffusion methods were common

phenotypic tests used for determining the AST of the isolates. Vitek, E-test and agar dilution

methods were less used. PCR or PCR-based typing methods such as multi-locus sequence

typing (MLST), repetitive element PCR (REP), and enterobacterial repetitive intergenic

consensus (ERIC)-PCR were more commonly used to determine the clonality of

Enterobacterial isolates than non-PCR-based techniques such as pulse-field gel

electrophoresis (PFGE), which is more laborious. Finally, PCR was the commonest tool used

for determining the ARGs of the isolates, with the use of whole-genome sequencing being

limited (Table 2).

Discussion

The resistome dynamics or epidemiology, phylogenomics and phylogeography of Gram-

negative bacterial species and associated clones, MGEs and ARGS in Africa are herein

[14] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

limned for the first time. We show that E. coli, K. pneumoniae, S. enterica, A. baumannii, P.

aeruginosa, N. meningitidis/gonorrhoeae, V. cholerae, S. marcescens, Enterobacter spp. C.

jejuni, Mycoplasma spp., Providencia spp., Proteus mirabilis and Citrobacter spp. are major

pathogenic species with rich and diverse resistomes and mobilomes, circulating in humans,

animals and environment in Southern, Eastern, Western and Northern Africa. The poorer

sanitary conditions, food insecurity as well as weaker healthcare and diagnostic laboratory

capacities in Africa are well-known, accounting for the higher infectious disease rates on the

continent 2–5,18,24. Subsequently, the combination of diverse ARGs in highly pathogenic

species with wide geographical distribution on the continent is a cause for concern as it

provides fertile breeding grounds for periodic and large outbreaks with untold morbidities

and mortalities 25–28.

Although E. coli was the most isolated species in human, animal and environmental

specimens, it did not harbour the most diverse and richest resistome repertoire. This is

interesting as E. coli is mostly used as a model and index organism to study Gram-negative

bacterial resistance epidemiology 18,29. Whereas the higher numbers of E. coli strains obtained

in Africa could be due to its easy cultivability, identification, and exchange of resistance

determinants 17,30, as well as its common use as a model and/or index organism18,29, its lower

resistome diversity and richness could mean it is not representative of the actual resistome

circulating in any niche at a given point in time. Hence, Enterobacter cloacae/spp. and K.

pneumoniae, which contained richer resistomes, could serve as better representatives and

reporters of prevailing ARGs in any niche at a point in time. Particularly, the richer

resistomes of these two species strongly suggest that they can easily exchange ARGs between

themselves and other species, as reported already 17,30–32.

The higher resistome diversity, as well as the high isolation rates, of K. pneumoniae and

Enterobacter spp. are not surprising. Specifically, K. pneumoniae is the most isolated clinical

[15] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

bacterial pathogen in many countries worldwide, found to be involved in many fatal and

multidrug resistant infections 25,28,33,34. International clones such as K. pneumoniae ST208

and ST101 are implicated in the clonal dissemination of carbapenemases as well as colistin

and multidrug resistance 25,28,33–36; although ST208 was absent in Africa, ST101 was common

in several countries. As well, Enterobacter spp. are increasingly being isolated in many

clinical infections in which they are found to as a major host of mcr colistin resistance genes

and other clinically important MDR determinants 34,37–39. Notwithstanding its lower resistome

diversity compared to Enterobacter spp. and K. pneumoniae, the E. coli isolates contained

important ARGs, such as mcr-1, blaNDM-1, blaOXA-48/181, and blaCTX-M-15 (Tables S1-S3), which

can be transferred to other intestinal pathogens 16,17,30. Notably, the E. coli isolates also

exhibited high resistance rates against important clinical antibiotics (Tables S1-S3). Finally,

the Enterobacter spp., K. pneumoniae, and E. coli strains were generally highly multi-clonal

and evolutionarily distant, suggesting little clonal dissemination (except E. coli ST103 and K.

pneumoniae ST101) of these species across the continent, albeit local and limited inter-

country outbreaks were observed (Fig. 2-3, S7-S8 & S17) 33,40.

S. enterica and C. coli/jejuni, which are important zoonotic and food-borne pathogens 24,41,

were found in animal/food, human and environmental specimens, although S. enterica was

more common and had a higher resistome diversity than C. coli/jejuni (Tables 1 & S1-S3;

Fig. 7, S9 & S15). Notably, S. Typhi, S. Typhimurium, S. Entiritidis and S. Bovismorbificans

were mostly isolated from humans, with some S. Entiritidis and S. Infantis strains being

isolated from both humans and cattle (Fig. 7 & S9). Indeed, reports of S. Typhimurium and S.

Entiritidis isolation from pigs and poultry respectively, as well as their implication in fatal

zoonotic infections through contaminated food animal consumption, are well-documented

24,42–45. S. Typhi, a common food-borne pathogen that infects millions of people worldwide

annually and results in typhoid fever, diarrhoea and death in severe cases 18,20,21,46, was the

[16] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

third most common species to be isolated and the fourth species to host the most resistome

repertoire. As shown in Tables S4-S6 and Fig. 7 & S9, several isolates from Eastern,

Southern and Western Africa shared the same clone (ST1, ST2) and clade, representing

clonal outbreaks affecting many people over a large swathe of Africa. Moreover, S. Typhi

and other S. enterica serovars recorded very high resistance rates to first-line antibiotics in

many African countries within the study period (Table S2). This is a very concerning

observation given the widespread and periodic incidence of outbreaks involving this

pathogen in most developing countries 18,21.

C. coli/jejuni strains were reported in substantial numbers from animals, which are their

natural hosts 47, as well as from human and environmental specimens, albeit few genomes (all

from South Africa, including C. concisus from human faeces) of these species were available

from the continent (Table 1; Fig. S15). As well, they were not as geographically distributed

as S. enterica as they were reported from only Botswana (human excreta and chicken

caecum), Cameroon (household water), South Africa (human excreta and river water), and

Tanzania (cattle milk/beef). Moreover, they harboured relatively fewer ARGs and had

generally lower resistance rates, albeit resistance to ampicillin, azithromycin, ciprofloxacin,

erythromycin, nalidixic acid, tetracycline, was high (Tables S1-S3). Interestingly, the C.

coli/jejuni isolates were mostly multiclonal, suggesting evolutionary versatility and

polyclonal dissemination. Campylobacter spp. are implicated in many diarrhoeal cases and

are the major cause of human bacterial gastroenteritis worldwide, causing fatal infections in

infants, the elderly and immunocompromised patients 47. Hence, the little data available on

this pathogen is disturbing as it makes it difficult to effectively plan appropriate interventions.

However, their presence in humans, animals and the environment in substantial numbers

shows their host adaptability, making them ideal candidates for One Health surveillance

studies.

[17] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

V. cholerae, another common food- and water-borne diarrhoea-causing pathogen implicated

in recurring outbreaks in many parts of Africa 13,48, was also reported in substantial numbers

from human and more importantly, from environmental sources in several countries (Table 1;

Fig. 8 & S2). The higher isolation of V. cholerae ST69 and ST515 clones across several

countries in Southern, Eastern, and Western African countries, which clustered within three

main clades having very close evolutionary distance and highly conserved but rich resistome

repertoire, shows the presence of same and highly similar strains (with little evolutionary

variation) circulating in Africa and causing recurring outbreaks with high morbidities and

mortalities. Just as S. Typhi and Campylobacter spp., V. cholerae also cause serious diarrhoea

in addition to vomiting in patients and has been implicated in death in untreated patients

within hours 13,48. Subsequently, the large ARGs diversity in these strains is concerning.

Indeed, a carbapenemase gene, termed blaVCC-1, mediating resistance to carbapenems and

most β-lactams, has been recently detected in Vibrio spp. 16,49,50, although this was not found

in any of these isolates.

Non-fermenting Gram-negative bacilli such as P. aeruginosa, A. baumannii, S. maltophilia

and A. hydrophilia are known opportunistic nosocomial pathogens with intrinsic resistance to

several antibiotics 51–55. Particularly, P. aeruginosa and A. baumannii, which were two of the

commonest pathogens with most ARGs in Africa, are commonly implicated in several

difficult-to-treat and fatal clinical infections worldwide 51–55. Thus, the higher resistome

diversity, geographical distribution, isolation and resistance rates of these pathogens are not

surprising. Whereas OXA-23/51-like carbapenemases are known to be common in A.

baumannii 34,56, the uniform presence of OXA-48-like carbapenemases in P. aeruginosa

genomes from Africa is very worrying, particularly given the wider geographical distribution

and ubiquity of this pathogen (Fig. 6)51,54. Owing to the broad β-lactams spectrum of

carbapenemases and the importance of β-lactams in treating bacterial infections, the presence

[18] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

of these and other ARGs in these pathogens with high intrinsic resistance in Africa is a cause

for concern 4,34,56. Given the difficulty in treating infections caused by non-fermenting Gram-

negative bacilli, it is quite refreshing to note that S. maltophilia and A. hydrophilia were less

isolated with little or no ARGS.

Important sexually transmitted infections such as gonorrhoea (N. gonorrhoeae) and non-

gonococcal urethritis (M. genitalium), respiratory infections such as pneumonia (M.

pneumoniae), cystic fibrosis (aggravated by B. cepacia) and whooping cough (B. pertussis),

as well as cerebro-spinal infections such as meningitis (N. meningitidis) are caused by GNB,

killing millions of people annually 6–15,57. Unfortunately, only N. meningitidis genomes were

reported from Africa (17 countries), although both N. meningitidis (Egypt and Niger

(serogroups C & W)) and N. gonorrhoeae (only Kenya) were found in the literature (Table

S6; Fig. 5). Notably, tet(B) was the sole ARG found in N. meningitidis genomes, particularly

clades B5, C and D, whereas several other mechanisms (gyrA, penA and rpoB) were reported

in the literature (Table S2). These differences in the literature and genomic resistomes is quite

interesting as some of the genomes were from Niger. Within each N. meningitidis clade were

isolates from different countries, suggesting inter-boundary transmission. This is not

surprising given the high transmissibility of N. meningitidis and the inter-country trade

existing within the Sahel and West African nations with the highest concentration of this

pathogen (Table S6; Fig. 5)8.

Worryingly, two N. meningitidis strains from Egypt were MDR to clinically important

antibiotics such as ciprofloxacin, cefotaxime, amikacin, ampicillin, penicillin and

meropenem. Sadly, reports of MDR N. gonorrhoeae that are highly resistant to first-line anti-

gonococcal drugs such as third-generation cephalosporins and azithromycin are increasing,

prompting revisions in treatment guidelines 6,57–59. For instance, MDR N. gonorrhoeae that

were only treatable with carbapenems have been reported in the UK and Australia 57–59.

[19] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Unfortunately, N. gonorrhoeae resistance rates were not calculable due the absence of N.

gonorrhoeae ASTs. Indeed, the use of molecular tests to determine N. gonorrhoeae

resist57ance, although fast, is unable to provide AST data that is critical to guide treatment.

The shift to molecular tests is thus making N. gonorrhoeae AST data scarce and could

account for the dearth of information on N. gonorrhoeae AST in the included articles57.

M. genitalium genomes from Africa were not found, although they have been reported in the

South Africa as having gyrA and parC resistance determinants (Table S2). However, M.

pneumoniae genomes, obtained from Southern, Western, Eastern and Northern Africa,

harboured no known ARGs. Other species such as M. gallinarum, M. gallinaceum, M.

pullorum, M. mycoides and M. capripneumoniae were found in animals whilst M. arginini

were isolated from the environment. Like Neisseria spp., increasing macrolide resistance in

Mycoplasma spp. are being reported, making them less sensitive to azithromycin.

Although B. cepacia does not cause cystic fibrosis, it aggravates it 10. B. cepacia are

inherently resistant to treatment by most antibiotics and they normally occur alongside P.

aeruginosa in cystic fibrotic lungs, where they can cause persistent infections and death 10,60.

In general, Burkholderia spp. were very few throughout Africa, in both literature and

published genomes, with no known resistance gene being reported (Fig. S14). B. pertussis,

the causative agent of whooping cough, a major childhood killer disease that has been killing

infants for thousands of years 14, was rarely reported in the literature and had fewer public

genomes from only Kenya and South Africa. In fact, the fewer reports of this infection in the

literature does not reflect the epidemiology of whooping cough on the continent as it kills

many infants in Africa yearly 61, suggesting that most clinical cases are not published.

Although vaccinations have reduced the mortality and morbidity rates associated with this

pathogen, variations in the pathogen is reducing the efficacy of the vaccine and increasing its

re-emergence globally 61.

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Whilst other Enterobacteriaceae (GNB) species such as S. marcescens, Citrobacter spp.,

Providencia spp., Proteus mirabilis, and M. morganni were relatively less isolated than E.

coli and K. pneumoniae, they did harbour a rich and diverse collection of resistomes in

different clones/clades across Africa, although many of these were mainly reported from

South Africa, followed by Tanzania, Nigeria, Senegal and Egypt (Fig. S16, S18, & S21-23).

These species have been implicated in fatal infections such as sepsis and wound infections

31,34,39,62,63, bearing critical ARGs such as ESBLs and carbapenemases (Table S2). On the

other hand, rarely isolated/reported GNB species such as Trabulsiella spp., Psychrobacter

spp., Myroides spp., Chromobacterium spp., Brevundimonas spp. and Bacillus spp. were

mainly obtained from the environment or termites in very few countries and harboured no

ARGs. As well A. faecalis, Leclercia adecarboxylata, Pantoea spp. and Raoultella spp.,

which were mainly isolated from human specimen except for Pantoea spp. that was also

obtained from animals and humans, also contained no known ARG and were mainly

restricted to few geographical areas except Pantoea spp. that was widely distributed across

Africa (Tables 1, S1-S3 & Fig. S11, S20, S24-S25).

Future perspectives & Conclusion

The backbone to efficient diagnosis and treatment of infections is rapid, effect, simple and

cheap diagnostics and skilled laboratory scientists 64–66. Without appropriate diagnostics, the

aetiology of many infectious diseases, the genotype/clone of the infecting organism and its

resistance mechanisms cannot be known, and a proper therapeutic choice cannot be made 64–

66. This describes the situation in many African countries, making several preventable

infections emerge subtly into large scale outbreaks 65,66. As shown in Table 2, simpler

phenotypic diagnostic tests with a longer turnaround time of at least 24 hours were more

commonly used in Africa whilst complex, skill-requiring and expensive tests like whole-

[21] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

genome sequencing and Vitek were hardly used. These challenges affect the fight against

infectious diseases and make surveillance studies on the continent difficult 64,67.

It is worthy of consideration that as large as the number of articles and genomes used in this

study are, the dearth of genome sequencing and molecular ARG surveillance in many African

countries, influenced by low funding, absence of molecular diagnostic laboratories, and

inadequate skilled personnel, affect the comprehensiveness of the phylogeography and

resistome evolutionary epidemiology 4. This is particularly true for certain species such as V.

cholerae, C. coli/jejuni, N. meningitidis/gonorrhoeae, Mycoplasma genitalium, Providencia

spp., M. morganii, C. freundii, P. mirabilis, B. cepacia, and B. pertussis, which are very

important clinical pathogens implicated in substantial morbidities and mortalities 19.

In summary, MDR GNB clinical pathogens implicated in high morbidities and mortalities are

circulating in Africa in single and multiple clones, shuttling diverse resistomes on plasmids,

integrons, insertion sequences and transposons from animals, foods, plants and the

environment to humans. A comprehensive One Health molecular surveillance is needed to

map the transmission routes and understand the resistance mechanism of these pathogens to

inform appropriate epidemiological interventions.

Funding: None

Author contributions: JOS conceived and designed the study, searched the literature,

analysed the data and undertook all bio-informatic analyses, image designs and tabulations

and wrote the paper. MAR searched the literature, extracted the data and resistance genes

from NCBI into Excel sheets and undertook frequency analyses of the raw data.

[22] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Transparency declaration: The authors declare no conflict of interest

Acknowledgements: We are exceptionally grateful to Dora Osei Sekyere of the University

of Education Winneba, Kumasi Campus, Ghana, for aiding in the curation, annotation and

design of the genomic and phylogenomic metadata and trees.

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Table 1. Species distribution frequencies per specimen sources Genera (species) Animal specimens Human specimens Environmental Total Genomes included in doi: (n = 5 950) (n = 57 464) specimens (n = 1 705) phylogenomics https://doi.org/10.1101/2020.04.09.20059766 Escherichia (coli, hermannii,vulneris) 1566 4899 2827 9292 592 Salmonella (Enteritidis, Paratyphi, Typhi, Typhimurium) 500 1262 11 487 1773 Klebsiella (pneumoniae, oxytoca, variicola, michiganensis) 204 2528 44 2776 311

Pseudomonas (aeruginosa, fluorerscens, otitidis, putida) 45 1366 87 1498 95 All rightsreserved.Noreuseallowedwithoutpermission. Acinetobacter (baumannii, calcoaceticus, haemolyticus) 2 611 92 705 21 Campylobacter (coli, jejuni) 210 175 105 490 13 Citrobacter (amalonaticus, freundii, koseri, farmer) 1 133 6 140 192 Enterobacter (aerogenes, amnigenus, cloacae, sakazakii) 21 459 0 480 60 Achromobacter xylosoxidans 1 0 0 1 0 ; Providencia (stuartii, rettgeri) 1 48 6 55 132 this versionpostedApril14,2020. Proteus (mirabilis, vulgaris) 0 394 53 447 159 Serratia (marcescens, liquefasciens, odorifera) 0 144 4 148 197 Burkholderia spp. 0 6 0 6 7 Vibrio cholerae 0 24 279 303 180 Morganella (morganii) 0 31 19 50 85 Neisseria (meningitidis, gonorrhoea) 0 207 0 207 199 Bordetella (bronchiseptica) 0 1 2 3 21 The copyrightholderforthispreprint Alcaligenes faecalis 0 3 47 50 0 Stenotrophomonas (maltophilia) 0 5 126 131 6 Leclercia adecarboxylata 0 1 0 1 0 Pantoea (dispersa, agglomerans) 0 2 1 3 21 Mycoplasma genitalium 0 266 0 266 23 Raoultella 0 3 0 3 0 Aeromonas 0 0 11 11 3 Bacillus 0 0 97 97 96 medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Brevundimonas 0 0 3 3 120 Chromobacterium 0 0 6 6 0

Myroides 0 0 2 2 0 doi:

Psychrobacter 0 0 3 3 0 https://doi.org/10.1101/2020.04.09.20059766 Trabulsiella 0 0 1 1 8 Total 2551 12568 3832 18951 3028 • Discrepancies between total isolates in Table 1 and total isolates under Results arise from the fact that not all isolates were Gram-negative isolates. Non-Gram-

negative isolates are not included in Table 1. All rightsreserved.Noreuseallowedwithoutpermission.

Table 2. Diagnostics used for detection, typing and resistance characterisation Diagnostic or typing tool/assay Animals Humans Environment

Phenotypic antibiotic sensitivity testing

Agar dilution method 1 0 3 ; this versionpostedApril14,2020. Disk diffusion method 19 35 7

Broth Micro-dilution MIC test 12 51 10

E-test 0 7 1

VITEK®2 System 0 19

Molecular typing

1 AFLP 1 0 0 The copyrightholderforthispreprint

MLST/cgMLST2 7 47 2

Whole-genome sequencing (WGS) 3 6 0

PCR‐based phylogenetic typing (ERIC-/ 9 11 3

1 Amplified fragment length polymorphism (AFLP) 2 Multi-locus sequence typing (MLST)/core-genome MLST (cgMLST) [2]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. REP-PCR)

PFGE/S1-PFGE3 3 18 1 doi: https://doi.org/10.1101/2020.04.09.20059766 Molecular detection of ARGs (antibiotic resistance genes)

PCR 27 92 19

WGS 5 23 1

Sanger sequencing 0 0 1 All rightsreserved.Noreuseallowedwithoutpermission.

Southern blotting 0 2 0

Figure 1. The PRISMA flow-diagram was used to summarise the literature search methodology, databases used, and the inclusion and exclusion ; this versionpostedApril14,2020. criteria adopted in getting the final 146 manuscripts used in this qualitative and quantitative analyses. Besides the included literature, 3028 publicly available genomes were downloaded from PATRIC (https://www.patricbrc.org/)/NCBI’s Genbank and analysed to determine their resistomes and evolutionary phylogeography.

Figure 2. Phylogeographic distribution of Acinetobacter baumannii clades and associated resistomes in Africa. The included A. baumannii genomes were mainly from Tunisia, Togo, Ghana, Sudan, and South Africa, with clade-specific ARGs; most of these strains were from humans. The copyrightholderforthispreprint

Cluster A, which was not A. baumannii, had no ARGs, whilst clusters/clades B1 and B2 had OXA-23-/66-like carbapenemases, ble, ant(2’’)-Id, aph(3’)-Ib and ant(3”)-IIa . Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

3 Pulsed-field gel electrophoresis (PFGE) [3]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure 3. Phylogeographic distribution of Escherichia coli clades and associated resistomes in Africa. The E. coli clades were mostly from humans, mainly distributed in West and East Africa, Egypt and South Africa. Relatively few were from the environment and animals. The ARGs doi: https://doi.org/10.1101/2020.04.09.20059766 (tet (A/B/C/D), blaTEM-1, sul, aph(3”)-Ib, aph(6)-Id, and dfrA) were mostly conserved across the various clades, which were not region-specific, but mixed up. Strains from humans shared very close phyletic relationship with strains from animals and plants. Inter-country as well as human- animal dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively All rightsreserved.Noreuseallowedwithoutpermission. coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure 4. Phylogeographic distribution of Klebsiella pneumoniae clades and associated resistomes in Africa. Klebsiella pneumoniae was mainly from humans, with a few being isolated from plants and animals. The various clades were mixed up in South Africa, Tanzania, Uganda, West ; and North Africa. Strains from humans shared very close phyletic relationship with strains from animals and plants. The clades harboured many this versionpostedApril14,2020. conserved ARGs (n=14): aac(3’)-Ia/IIa, aac(6’)-IIa/Ib-cr, aph(3”)-Ib, aph(6’)-Id, blaCTX-M, blaOXA, blaSHV, blaTEM, aadA, catA/B, dfrA, fosA, oqxAB, sul1/2. Other ARGs that were substantially found in K. pneumoniae included arr, mph(A/E), qnrA/B/S, qacEΔ1 and blaNDM-1/5. A few genes were restricted to certain clades. Inter-country as well as human-animal dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. The copyrightholderforthispreprint

Figure 5. Phylogeographic distribution of Neisseria meningitidis clades and associated resistomes in Africa. Neisseria meningitidis was only from human samples from South Africa, Comoros, DRC, CAR, Cameroon, West Africa, Djibouti and Algeria; however, N. meningitidis was most concentrated in West Africa and the ARG (only tet(B)) was only found in clades B5, C and D. Inter-country dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. [4]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure 6. Phylogeographic distribution of Pseudomonas spp. clades and associated resistomes in Africa. Pseudomonas spp. were widely distributed in Africa, particularly Southern and Eastern Africa, DRC, West and (Cameroon, Nigeria, Benin, Ghana) North (Morocco, Algeria, doi:

Tunisia and Egypt) Africa. The other Pseudomonas spp. were mainly found in the environment and plants with no ARGs whilst P. aeruginosa https://doi.org/10.1101/2020.04.09.20059766 were found in humans and the environment with aph(3’)-IIb, blaOXA, blaPDC, catB7 and fosA being largely conserved in this pathogen. Clade B had additionally conserved genes such as sul1/2, and qacL/ΔE1. Inter-country as well as human-environment dissemination of isolates of the All rightsreserved.Noreuseallowedwithoutpermission. same clade were observed.Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure 7. Phylogeographic distribution of Salmonella enterica clades and associated resistomes in Africa. Salmonella enterica serovars were ; mainly host-specific, with S. Typhi, S. Typhimurium, S. Entiritidis, and S. Bovismorbificans being isolated from humans whilst serovars such as this versionpostedApril14,2020.

S. Muenster, S. Legon, NGUA, S. Salamae, and S. Wilhelmsburg strains were animal-associated. In general, S. enterica clades were of diverse phylogeographic distribution, but clustered in Southern Africa, Madagascar, DRC, Sudan, Comoros, Cameroon, East, West and North Africa.

The ARGs in S. enterica were serovar and clade-specific, with S. Typhi, S. Typhimurium, S. enterica NGUA strains hosting most ARGs such as

TEM, tet(A/B/C/D), aph(6’)-Id, aph(3”)-Ib, catA/B, dfrA, qacL/ΔE1, and sul1/2/3; notably, S. Typhi clades A1, B1-B3 (Fig. S9A) mostly The copyrightholderforthispreprint harboured these ARGS. Inter-country as well as human-animal-environment dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure 8. Phylogeographic distribution of Vibrio cholerae clades and associated resistomes in Africa. V. cholerae was only reported from humans in Southern and Eastern Africa, with few from Cameroon, Guinea, Sudan and Egypt. Members of clade B, which were very closely clustered together with little evolutionary variations, were mainly located in Kenya, Mozambique and Tanzania. Clades A and C were more [5]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. geographically diverse with more evolutionary variations. As supported by the resistome data, clade B had almost uniform resistomes throughout, followed closely by clade C and A, which had the least repertoire of resistomes. Conserved within clades B and C were aph(3”)-Ib, doi: aph(6’)-Id, catB9, dfrA1, floR, sul1/2/3 and varG. Other ARGs in clade B were TEM-63 and tet(A/D/G); aadA1/2 and qacL/ΔE1 were only https://doi.org/10.1101/2020.04.09.20059766 found in clade A. Inter-country dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. All rightsreserved.Noreuseallowedwithoutpermission.

Figure S1. Species distribution and frequencies per animal, human and environmental sources in respective Africa countries. The major pathogens of medical importance found in Africa included E. coli, K. pneumoniae, S. enterica, P. aeruginosa, A. baumannii, Campylobacter coli/jejuni, Proteus mirabilis, Enterobacter spp., N. gonorrhoea/meningitidis and V. cholerae, and these were mainly found in humans, followed ; by animals and the environment. These were mainly reported from Algeria, Burkina Faso, Egypt, Ghana, Kenya, Libya, South Africa, Tanzania this versionpostedApril14,2020. and Tunisia in humans. South Africa, Tanzania and Nigeria, reported the highest concentrations of environmental species. Notably, E. coli and S. enterica, and to a lesser extent Campylobacter coli/jejuni, Klebsiella spp., and Pseudomonas spp., were the most isolated species from animals in the reporting countries

Figure S2. Frequency distribution of Gram-negative bacterial species isolated from human, animal and environmental specimens and their The copyrightholderforthispreprint associated antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). E. coli was the most isolated species from animals, humans and the environment. There were more species from human sources than animal and environmental specimens, with Klebsiella pneumoniae,

Salmonella enterica, Pseudomonas aeruginosa and Acinetobacter baumannii being also common in human samples. CTX-M, TEM, OXA,

SHV, NDM, AAC(6’), AAD and QNR enzymes were common resistance mechanisms in clinical isolates whilst TET, SUL, TEM, CTX-M and

CMY were common resistance mechanisms in animal isolates; TET and TEM resistance mechanisms were more isolated in environmental [6]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. samples. There were more MGEs characterisation in human isolates than animal and environmental strains, with Class 1 integrons and

IncF/IncX plasmids being very common in human isolates. doi: https://doi.org/10.1101/2020.04.09.20059766 Figure S3. Clonal distribution frequency of Gram-negative bacterial species isolated from human, animal and environmental specimens. The clonality or genotypes of E. coli, K. pneumoniae, A. baumannii, Campylobacter coli/jejuni, V. cholerae, S. marcescens, N.

gonorrheae/meningitidis, S. enterica Enteritidis/Typhimurium were the main data reported by the included countries. Common clones such as E. All rightsreserved.Noreuseallowedwithoutpermission. coli ST131, ST38, ST410, E. coli group A/B/D (in Egypt and Algeria) were found in humans and to some extent, in animals and the environment. Clones of A. baumannii (ST1, ST2, ST218, ST208, ST415, ST493, ST441, ST499 and ST600 in Egypt and ST106, ST208, ST229,

ST339, ST502, ST758, ST848 and ST1552 in South Africa ), K. pneumoniae (ST15, ST39, ST152, ST154, ST101, ST147, ST466, ST2094), V. ; cholerae ST69 (Cameroon), P. aeruginosa (ST235 & ST233 in Egypt and ST244 & ST640 in Tanzania), S. marcescens types A & B (Egypt), N. this versionpostedApril14,2020. gonnorhoea (ST1599, ST1903, ST1893 & ST11366 in Kenya), N. meningitidis (ST11 & ST10217 in Niger), S. Typhimurium ST313 and S.

Enteritidis ST11 (Kenya) and S. Typhi ST1 (Zambia) were very common in humans.

Figure S4. ARGs reported from animals, humans and environmental Gram-negative bacterial isolates from the included articles per country.

The frequency distribution of the various ARGs that were isolated from the Gram-negative bacterial species shows that CTX-M, TEM, SHV, The copyrightholderforthispreprint

OXA, QnrA/B/D/S, AAC(6’)-Ib-cr, AAC(3’)-IIa, SUL/DFRA enzymes were common in selected countries/regions. In all, mechanisms mediating resistance to β-lactamases were common in all countries followed by fluoroquinolones, sulphamethoxazole-trimethoprim, aminoglyclosides, tetracycline, chloramphenicol, and erythromycin. Tetracycline resistance mechanisms were more common in animal isolates than human and environmental isolates; however, sulphamethoxazole-trimethoprim resistance genes were more common than those of tetracycline (tet genes). [7]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure S5. Mobile genetic elements (MGEs) found in species isolated from animals, humans, and the environment in Africa.

Figure S6. Mobile genetic elements (MGEs) and associated antibiotic resistance genes (ARGs) found in species isolated from animals, humans, doi: https://doi.org/10.1101/2020.04.09.20059766 and the environment.

Figure S7. Phylogeographic distribution of Escherichia coli clades and associated resistomes in Africa (A & B). The E. coli clades were mostly from humans, mainly distributed in West and East Africa, Egypt and South Africa. Relatively few were from the environment and animals. The All rightsreserved.Noreuseallowedwithoutpermission.

ARGs (tet (A/B/C/D), blaTEM-1, sul, aph(3”)-Ib, aph(6)-Id, and dfrA) were mostly conserved across the various clades, which were not region- specific, but mixed up. Inter-country as well as human-animal-environment dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. ; this versionpostedApril14,2020.

Figure S8. Phylogeographic distribution of Klebsiella pneumoniae clades and associated resistomes in Africa. Klebsiella pneumoniae was mainly from humans, with a few being isolated from plants and animals. The various clades were mixed up in South Africa, Tanzania, Uganda,

West and North Africa. Strains from humans shared very close phyletic relationship with strains from animals and plants. The clades harboured many conserved ARGs (n=14): aac(3’)-Ia/IIa, aac(6’)-IIa/Ib-cr, aph(3”)-Ib, aph(6’)-Id, blaCTX-M, blaOXA, blaSHV, blaTEM, aadA, catA/B, dfrA,

fosA, oqxAB, sul1/2. Other ARGs that were substantially found in K. pneumoniae included arr, mph(A/E), qnrA/B/S, qacEΔ1 and blaNDM-1/5. A The copyrightholderforthispreprint few genes were restricted to certain clades. Inter-country as well as human-food-environment dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

[8]

medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure S9. Phylogeographic distribution of Salmonella enterica clades and associated resistomes in Africa (A & B). Salmonella enterica serovars were mainly host-specific, with S. Typhi, S. Typhimurium, S. Entiritidis, and S. Bovismorbificans being isolated from humans whilst doi: serovars such as S. Muenster, S. Legon, NGUA, S. Salamae, and S. Wilhelmsburg strains were animal-associated. In general, S. enterica clades https://doi.org/10.1101/2020.04.09.20059766 were of diverse phylogeographic distribution, but clustered in Southern Africa, Madagascar, DRC, Sudan, Comoros, Cameroon, East, West and

North Africa. The ARGs in S. enterica were serovar and clade-specific, with S. Typhi, S. Typhimurium, S. enterica NGUA strains hosting most All rightsreserved.Noreuseallowedwithoutpermission. ARGs such as TEM, tet(A/B/C/D), aph(6’)-Id, aph(3”)-Ib, catA/B, dfrA, qacL/ΔE1, and sul1/2/3; notably, S. Typhi clades A1, B1-B3 (Fig. S9A) mostly harboured these ARGS. Inter-country dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. ; Figure S10. Phylogeographic distribution of Aeromonas spp. clades and associated resistomes in Africa. No known ARGs could be found in the this versionpostedApril14,2020. genomes, which were all from South African environmental specimens. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S11. Phylogeographic distribution of Bacillus spp. clades and associated resistomes in Africa. There were five main clusters, comprising various species of this genus, which were from animals (e.g. B. anthracis, B. cereus, B. thuringensis), humans (B. anthracis) environment (e.g. The copyrightholderforthispreprint

B. anthracis, B. cereus, B. thuringensis) and plants (e.g. B. subtilis, B. halotolerans, B. velenzensis) in Southern Africa, DRC, Kenya, Cameroon,

B. Faso, Senegal, and North Africa. No known ARGs could be found in the genomes. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

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medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure 12. Phylogeographic distribution of Bordetella spp. clades and associated resistomes in Africa. The genomes were obtained from only

Kenya from humans (B. pertussis) or the environment (other species). No known ARGs could be found in the genomes. Isolates from humans, doi: animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. https://doi.org/10.1101/2020.04.09.20059766

Figure S13. Phylogeographic distribution of Brevundimonas spp. clades and associated resistomes in Africa. Only a single strain from South

Africa was reported throughout Africa. No known ARGs could be found in the genomes. Isolates from humans, animals, the environment and All rightsreserved.Noreuseallowedwithoutpermission. plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S14. Phylogeographic distribution of Burkholderia spp. clades and associated resistomes in Africa. Burkholderia spp. genomes in Africa were solely from South Africa and B. Faso in plants and environmental samples. B. cepacia genomes were not found in Africa and no known ; this versionpostedApril14,2020. ARGs could be found in the included genomes. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S15. Phylogeographic distribution of Campylobacter spp. clades and associated resistomes in Africa. The included genomes were all from human (C. concisus/jejuni) and animal (C. fetus) specimens from South Africa with no known ARGs in the genomes. Isolates from

humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. The copyrightholderforthispreprint

Figure S16. Phylogeographic distribution of Citrobacter spp. clades and associated resistomes in Africa. The Citrobacter spp. genomes were from humans and water in South Africa, Tanzania, Nigeria, and Senegal. blaCMY was the only ARG conserved in all the clades whilst clade I had more ARGs; generally, the ARGs were not clade specific, but isolate-specific. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

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medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure S17. Phylogeographic distribution of Enterobacter spp. clades and associated resistomes in Africa. E. aerogenes, E. asburiae, E. cloacae, E. hormaechei, and E. xiangfangensis were obtained from animals, humans, environment (water) and plants in mainly South Africa, doi:

Zambia, West and North Africa. The Enterobacter spp. had a rich resistome repertoire (n≥25), comprising of ARGs such as aac(3’)-IIa, aac(6’)- https://doi.org/10.1101/2020.04.09.20059766

IIa/Ib3/-IIc/Ib-cr, aph(3”)-Ib, aph(3’)-I/Ia, aph(6’)-Id, blaACT, blaCMH(clade A only), blaNDM, blaCTX-M, blaOXA, blaOXA-48/181, blaSHV, blaTEM, aadA, catA/B, dfrA, fosA, oqxAB, sul1/2, qnrB/S, tet(A/D), mcr-1/9, arr, and rmtC. Inter-country as well as human-animal-water-food All rightsreserved.Noreuseallowedwithoutpermission. dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S18. Phylogeographic distribution of Morganella morgannii clades and associated resistomes in Africa. Only two isolates from humans ; in South Africa were obtained for all of Africa; one of these two strains had a relatively rich resistome. Isolates from humans, animals, the this versionpostedApril14,2020. environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S19. Phylogeographic distribution of Mycoplasma spp. clades and associated resistomes in Africa. These strains were host-specific, with

M. gallinarum/pullorum/gallinaceum/mycoides/capripneumoniae being found in animals and M. pneumoniae being found in humans; M. arginini was found in the environment. These strains were mainly found in Egypt, Cameroon, Nigeria, Tunisia, Southern and Eastern Africa. No The copyrightholderforthispreprint known ARGs could be found in the genomes. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

Figure S20. Phylogeographic distribution of Pantoea spp. clades and associated resistomes in Africa. Pantoea spp. were mainly found in the environment and in termites in South Africa, Tanzania, Rwanda, Uganda, West Africa, Algeria and Tunisia. No known ARGs could be found in

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medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. the genomes. Inter-country as well as plant-animal (termite) dissemination of isolates of the same clade were observed. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. doi: https://doi.org/10.1101/2020.04.09.20059766 Figure S21. Phylogeographic distribution of Proteus mirabilis clades and associated resistomes in Africa. P. mirabilis genomes were only found in human samples from Egypt and South Africa, with substantial differences in their resistomes. Isolates from humans, animals, the

environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. All rightsreserved.Noreuseallowedwithoutpermission.

Figure S22. Phylogeographic distribution of Providencia rettgerii clades and associated resistomes in Africa. All the P. rettgerii strains were from South Africa and contained several ARGs. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. ; this versionpostedApril14,2020.

Figure S23. Phylogeographic distribution of Serratia marcescens clades and associated resistomes in Africa. The S. marcescens strains were from mostly humans, with one being from a nematode; they were all isolated from South Africa. NDM-1 was common in the resistomes of all the isolates, with CTX-M-3, TEM and OXA-1 ESBLs being very common in many of the strains. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. The copyrightholderforthispreprint Figure S24. Phylogeographic distribution of Stenotrophomonas maltophilia/rhizophila. clades and associated resistomes in Africa. These two species were found in humans, water/environment and plants (S. rhizophila omly) in South Africa, DRC, Nigeria, Algeria and Morocco, with no known ARGs in the genomes. Isolates from humans, animals, the environment and plants are respectively coloured as blue, red, mauve/pink and green on the phylogeny tree.

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medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Figure S25. Phylogeographic distribution of Trabusiella spp. clades and associated resistomes in Africa. These genomes were mainly isolated from termites in South Africa, with o known ARGs found in the genomes. Isolates from humans, animals, the environment and plants are doi: respectively coloured as blue, red, mauve/pink and green on the phylogeny tree. https://doi.org/10.1101/2020.04.09.20059766

Supplementary Table S1. An Excel Table showing the general extracted meta-data from the included articles for animal samples. This sheet

contains all the information on bacterial species, clones, resistance mechanisms and associated mobile genetic elements (MGEs), and resistance All rightsreserved.Noreuseallowedwithoutpermission. rate per antibiotic for each species. Resistance rates above 50% are shown in red for easy identification by the reader. Subsequent downstream qualitative and quantitative analyses were undertaken from these Tables.

Supplementary Table S2. An Excel Table showing the general extracted meta-data from the included articles for human samples. This sheet ; this versionpostedApril14,2020. contains all the information on bacterial species, clones, resistance mechanisms and associated mobile genetic elements (MGEs), and resistance rate per antibiotic for each species. Resistance rates above 50% are shown in red for easy identification by the reader. Subsequent downstream qualitative and quantitative analyses were undertaken from these Tables.

Supplementary Table S3. An Excel Table showing the general extracted meta-data from the included articles for environmental samples. This

sheet contains all the information on bacterial species, clones, resistance mechanisms and associated mobile genetic elements (MGEs), and The copyrightholderforthispreprint resistance rate per antibiotic for each species. Resistance rates above 50% are shown in red for easy identification by the reader. Subsequent downstream qualitative and quantitative analyses were undertaken from these Tables.

Supplementary Table S4. An Excel Table showing the metadata of all strains used for the genomic, resistome and phylogenetic analyses. Each species is placed on a single sheet within the file and contains all genomic and phenotypic information on the strains.

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medRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaypreprintinperpetuity. Supplementary Table S5. Pictorial representation of the resistomes of all strains per species. The resistomes of each strain per species is found within separate sheets. The clades and subclades of each species is coloured differently to show similarities and evolutionary dynamics across doi: countries. Members of the same clade are coloured with the same colours irrespective of their MLST. https://doi.org/10.1101/2020.04.09.20059766

Supplementary Table S6. Species by species breakdown of the resistomes of each included genome. The resistomes and associated genomic

and demographic strain data are shown in this file without colour codings. All rightsreserved.Noreuseallowedwithoutpermission.

; this versionpostedApril14,2020. The copyrightholderforthispreprint

[14] medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. medRxiv preprint doi: https://doi.org/10.1101/2020.04.09.20059766; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.